Compositions and kits for herpes simplex virus type 1 and 2 nucleic acid detection

ABSTRACT

The invention relates to polynucleotides for HSV detection and the use of these polypeptides in kits and methods for HSV detection.

FIELD OF THE INVENTION

The present invention relates to novel polynucleotides, molecularbeacons, methods and kits for the detection of herpes simplex virustypes 1 and 2.

BACKGROUND OF THE INVENTION

Herpes Simplex Virus (HSV) type-1 and type-2 are human pathogens thatcause primary, latent, and recurrent infections. HSV infection hasvarious clinical manifestations including genital herpes, oral-facialinfections, cutaneous and ocular infections, herpes encephalitis,neonatal herpes and erythema multiforme. HSV-1 predominantly causesherpes labialis and HSV-2 causes herpes vulvovaginitis. Genital HSVinfection is a sexually transmitted disease that is prevalent worldwideand is becoming an increasingly important public health issue. There hasbeen a 30% increase in the incidence of sexually transmitted HSVinfections over the last two decades in the United States alone.Detection of HSV during pregnancy is very important since exposure ofthe neonate during delivery could result in infection. HSV infection iscommonly treated using anti-viral agents such as acyclovir, valacycloviror famciclovir.

Common methods of detection of HSV infection include serologicaltesting, viral culture or polymerase chain reaction (PCR) followed bymicrotiter plate detection (Riley, 1998). These assays are timeconsuming and are not amenable to the rapid diagnosis and treatment ofHSV infected individuals. An assay which is faster, less labor intensiveand comparable in sensitivity to microtiter plate based assays isdesirable.

SUMMARY OF THE INVENTION

The polynucleotides, kits and methods of the present invention areutilized in a qualitative PCR assay for the detection of HSV in asample. Additionally, the assay can be performed in the presence of aninternal amplification control to detect false negative results. Themethods of the present invention require minimal manipulation and may beused and performed in a closed tube, thereby greatly reducing the riskof contamination and reducing the time and effort needed for the test.

The invention provides for a purified polynucleotide selected from thegroup consisting of SEQ ID NOS:1-11.

The invention also provides for a pair of polynucleotide primers for apolymerase chain reaction, wherein the primers comprise SEQ ID NO:1 andSEQ ID NO:2.

The invention also provides for a polynucleotide for HSV detection,wherein said polynucleotide comprises SEQ ID NO:4.

In a preferred embodiment, the polynucleotide comprises a labeledpolynucleotide.

In another embodiment, the labeled polynucleotide comprises a pair offluorophore/quencher labels.

The invention also provides for a pair of polynucleotides for HSVdetection wherein said pair of polynucleotides is selected from thegroup consisting of SEQ ID NO:3 and SEQ ID NO:4, and SEQ ID NO:3 and,SEQ ID NO:3

In a preferred embodiment, the pair of polynucleotides comprises alabeled first polynucleotide and a labeled second polynucleotide,wherein the first and second polynucleotides are differentially labeled.

In another embodiment, the first and second differentially labeledpolynucleotides each comprises a pair of fluorophore/quencher labels.

In another embodiment, the fluorophore label is different between saidfirst and second polynucleotides and wherein said quencher label is thesame between first and second polynucleotides.

The invention also provides for a kit for HSV detection comprising apair of polynucleotides wherein said pair of polynucleotides is selectedfrom the group consisting of SEQ ID NO:3 and SEQ ID NO:4, and SEQ IDNO:3 and, SEQ ID NO:3, and packaging materials therefor.

The polynucleotide primers will include a forward and a reverse primer,the forward primer being complementary to a first strand of the targetnucleic acid and positioned upstream of (5′ to) a region in the targetsequence to be amplified, and the reverse primer will be complementaryto the second strand (or the complementary strand of the first strand)and positioned downstream of (3′ to) a region to be amplified.

In a preferred embodiment, the kit further comprises a pair ofpolynucleotide primers wherein the primers comprise SEQ ID NO:1 and SEQID NO:2, and a DNA polymerase.

The invention also provides for a kit for performing a polymerase chainreaction comprising a pair of polynucleotide primers wherein the primerscomprise SEQ ID NO:1 and SEQ ID NO:2, a DNA polymerase, and packagingmaterials therefor.

In a preferred embodiment, the DNA polymerase is thermostable.

In another embodiment, the kit further comprises a buffer suitable forHSV detection and polymerase chain reaction.

In another embodiment, the kit further comprises an internalamplification control plasmid comprising sequences presented in SEQ IDNO:8 AND SEQ ID NO:9.

In another embodiment, the kit further comprises a first controltemplate having a sequence presented in SEQ ID NO:6 and a second controltemplate having a sequence presented in SEQ ID NO:7.

The invention also provides for a kit for HSV detection, comprising apolynucleotide for HSV detection having a sequence presented in SEQ IDNO:4, a pair of polynucleotides for polymerase chain reaction wherein afirst polynucleotide of the pair has the sequence presented in SEQ IDNO:1 and a second polynucleotide of the pair has the sequence presentedin SEQ ID NO:2, a DNA polymerase, and a buffer suitable for HSVdetection and polymerase chain reaction.

In a preferred embodiment, the kit further comprises a controlpolynucleotide having the sequence presented in SEQ ID NO:3, and an IACplasmid comprising sequences presented in SEQ ID NO:8 AND SEQ ID NO:9.

In another embodiment, the kit further comprises a first controltemplate having a sequence presented in SEQ ID NO:6 and a second controltemplate having a sequence presented in SEQ ID NO:7.

The invention also provides for a kit for HSV detection, comprising apair of polynucleotides for HSV detection selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO 4, or SEQ ID NO:3 and SEQ IDNO:3, a pair of polynucleotides for polymerase chain reaction wherein afirst polynucleotide of the pair for polymerase chain reaction has thesequence presented in SEQ ID NO:1 and a second polynucleotide of thepair for polymerase chain reaction has the sequence presented in SEQ IDNO:2, a DNA polymerase, and a buffer suitable for HSV detection andpolymerase chain reaction.

In a preferred embodiment, the kit further comprises a controlpolynucleotide having the sequence presented in SEQ ID NO:3, and an IACplasmid comprising sequences presented in SEQ ID NO:8 AND SEQ ID NO:9

In another embodiment, the kit further comprises a first controltemplate having the sequence presented in SEQ ID NO:6 and a secondcontrol template having the sequence presented in SEQ ID NO:7.

The invention also provides for a method for HSV detection, comprisingthe steps of: (a) contacting a target nucleic acid with a polynucleotidecomprising SEQ ID NO:4, wherein the target nucleic acid comprises asequence complementary to the polynucleotide, wherein a hybrid formsbetween the target nucleic acid and the polynucleotide under conditionswhich permit formation of said hybrid; and (b) detecting said hybrid.

In a preferred embodiment, the polynucleotide is labeled.

In another embodiment, the detecting step comprises detecting emissionof fluorescence.

The invention also provides for a method for HSV detection, comprisingthe steps of: (a) mixing a target nucleic acid with a polynucleotide fordetecting HSV comprising SEQ ID NO:4, and a pair of polynucleotides forpolymerase chain reaction comprising SEQ ID NO:1 and SEQ ID NO:2,wherein the target nucleic acid comprises the sequence complementary tothe polynucleotide for detecting HSV and a sequence complementary to thepair of polynucleotides for polymerase chain reaction; (b) incubating amixture of step (a) under conditions which permit a polymerase chainreaction to generate a product comprising a sequence to thepolynucleotide for detecting HSV and which permit formation of a hybridbetween the polynucleotide for detecting HSV and said product; and (c)detecting the hybrid.

In a preferred embodiment, the polynucleotide for detecting HSV islabeled.

In another embodiment, the detecting step comprises detecting emissionof fluorescence.

The invention also provides for a method for HSV detection, comprisingthe steps of: (a) contacting a target nucleic acid with a pair ofpolynucleotides selected from the group consisting of SEQ ID NO:3 andSEQ ID NO:4 or SEQ ID NO:3 and SEQ ID NO:3, wherein the target nucleicacid comprises a sequence complementary to at least one of saidpolynucleotide, wherein a hybrid forms between the target nucleic acidand at least one of the polynucleotide under conditions which permitformation of the hybrid; and (b) detecting the hybrid.

In a preferred embodiment, the polynucleotides for detecting HSV aredifferentially labeled.

In another embodiment, the detecting step comprises detecting emissionof fluorescence.

The invention also provides for a method for HSV detection, comprisingthe steps of: (a) mixing a target nucleic acid with a pair ofpolynucleotides for detecting HSV selected from the group consisting ofSEQ ID NO:3 and SEQ ID NO:4 or SEQ ID NO:3 and SEQ ID NO:3, and a pairof polynucleotides for polymerase chain reaction comprising SEQ ID NO:1and SEQ ID NO:2, wherein the target nucleic acid comprises a sequencecomplementary to at least one of the polynucleotides for detecting HSVand a sequence complementary to the pair of polynucleotides forpolymerase chain reaction; (b) incubating a mixture of step (a) underconditions which permit a polymerase chain reaction to generate aproduct comprising a sequence of at least one of the polynucleotides fordetecting HSV and which permit formation of a hybrid between at leastone of the polynucleotide for detecting HSV and the product; and (c)detecting the hybrid.

In a preferred embodiment, the polynucleotides for detecting HSV aredifferentially labeled.

In another embodiment, the detecting step comprises detecting emissionof fluorescence.

Any of the kits described above may also include an internalamplification control (IAC). The internal amplification control providesa polynucleotide for hybridization to an IAC sequence, a control plasmidcomprising an IAC sequence, and a control single-strandedoligonucleotide comprising an IAC sequence. Preferably, the IACpolynucleotide comprises a sequence presented in SEQ ID NO: 3, the IACplasmid comprises a double-stranded IAC sequence synthesized by theextension of overlapping polynucleotides presented by SEQ ID NO:8 andSEQ ID NO:9, and the control single-stranded oligonucleotide IACsequence comprises a sequence presented in SEQ ID NO:5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences amplified by the PCR reaction for HSV-1 (SEQID NO: 12), HSV-2 (SEQ ID NO: 13) and IAC (SEQ ID NO: 14). Thenucleotides in caps (T or C) indicate the sequence difference betweenHSV-1 and HSV-2 and the positions where the HSV-TC molecular beaconhybridizes (underlined). Other highlighted nucleotides indicate othersequence differences between HSV-1 and HSV-2. The amplification primerbinding sites are italicized. The PCR products generated are 109 bp.

FIG. 2 is a photograph of PCR products electrophoresed on a 2% agaroseTBE gel to determine the optimal annealing temperature for HSV-TCamplification. The PCR product is 109 bp and indicated with an arrow.

FIG. 3 shows melting curves of the HSV-TC and IAC specific molecularbeacons.

FIG. 4 shows the results of a PCR analysis using the molecular beacon290 (FAM) for IAC and molecular beacon 294 (TET) for HSV-TC.

FIG. 4A shows the results when IAC template (0.5 pg/reaction) wasevaluated with b294 (TET) and b290 (FAM).

FIG. 4B shows HSV-1 viral DNA at two concentrations (5000 copies and 500copies) used as the template with b294 (TET).

FIG. 4C is the same as FIG. 4B except that HSV-2 viral DNA was used asthe template.

FIG. 4D shows the results when the template included 5000 copies ofHSV-1+IAC (0.5 pg/reaction) or 5000 copies of HSV-2+IAC (0.5pg/reaction) or no template (NTC) and evaluated with both b290 (FAM,IAC-specific) and b294 (TET, HSV-TC).

FIG. 5 shows the results of a PCR analysis used to determine thesensitivity of the HSV-TC molecular beacon.

FIG. 5A shows the results when HSV-1 was used as a template and

FIG. 5B shows the results when HSV-2 was used as a template. Bothreactions contained the HSV-TC and IAC molecular beacons.

FIG. 6 shows the results of clinical samples using the PCR-molecularbeacon assay of the present invention.

FIG. 7 shows the results of the PCR-molecular beacon assay using CSFspecimens when Method 3 is employed for the preparation of thespecimens. FIG. 7A shows the TET and FAM views while FIG. 7B shows thePCR products electrophoresed on a 2% agarose TBE gel.

FIG. 8 shows the results of the PCR-molecular beacon assay using CSFspecimens when Method 3A is employed for the preparation of thespecimens. The samples used are the same as those used in FIG. 7.

FIG. 8A shows the TET and FAM views while

FIG. 8B shows the PCR products electrophoresed on a 2% agarose TBE gel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a qualitative PCR assay for thedetection of HSV strains type-1 and type-2. The present inventionprovides a pair of polynucleotides for polymerase chain reaction (PCR)to amplify a HSV sequence present in a sample. The present inventionalso provides polynucleotides, preferably labeled with a fluorophore,for detecting both HSV viral strains. More particularly, thepolynucleotides for detecting HSV are molecular beacons. The presentinvention also provides polynucleotides, including templates andmolecular beacons, for internal amplification control reactions todetect false positives and false negatives. According to the presentinvention, the PCR, detection for HSV, and internal amplificationcontrols can be performed in the same reaction tube, thus reducing cost,time, and contamination. The present invention consistently allows thedetection of as few as 100 copies of HSV DNA per reaction.

Definitions

As used herein, the term “polynucleotide(s)” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. “Polynucleotide(s)” include, withoutlimitation, single- and double-stranded nucleic acids. As used herein,the term “polynucleotide(s)” also includes DNAs or RNAs as describedabove that contain one or more modified bases. Thus, DNAs or RNAs withbackbones modified for stability or for other reasons are“polynucleotide(s)”. The term “polynucleotide(s)” as it is employedherein embraces such chemically, enzymatically or metabolically modifiedforms of polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including, for example, simple andcomplex cells. “Polynucleotide(s)” also embraces short polynucleotidesoften referred to as oligonucleotide(s).

The term “molecular beacon” as used herein are single-strandedpolynucleotide probes that possess a stem-and-loop hairpin structure.The loop portion of the molecule is a probe sequence complementary to atarget sequence (e.g., an internal region of a sequence amplified byPCR) and the stem is formed by short complementary sequences located atthe opposite ends of the molecule. The molecule is labeled with afluorophore at one end and a quencher at the other end. When free insolution, the stem keeps the fluorophore and the quencher in closeproximity, causing the fluorescence of the fluorophore to be quenched byenergy transfer. When bound to its complementary target, theprobe-target hybrid forces the stem to unwind, separating thefluorophore from the quencher, and restoring the fluorescence. Thehairpin stem significantly enhances the specificity of molecularbeacons, enabling them to distinguish targets that differ by as littleas a single nucleotide. In addition, the hairpin conformation allows avariety of fluorophores to be used in conjunction with the samequencher. Thus, more than one molecular beacon, each labeled with adifferent fluorophore, can be used to detect several different targetsequences present in the same solution.

“HSV type common (TC)” as used herein refers generally to both HSVtype-1 and type-2 strains. The HSV type-1 and type-2 strains possesssignificant sequence identity and therefore, a single polynucleotide ormolecular beacon may be capable of hybridizing to both strains.

“HSV type specific” as used herein refers to the specific type of HSVstrain being detected. Although the HSV type-1 and type-2 possesssignificant sequence identity, a polynucleotide or molecular beacon canbe designed to hybridize to only one specific strain type, therebyallowing for discrimination between the two strains.

“Complementary” as used herein refers to the ability of a nucleic acidsingle strand (or portion thereof) to hybridize to an anti-parallelnucleic acid single strand (or portion thereof) by contiguousbase-pairing between the nucleotides (that is not interrupted by anyunpaired nucleotides) of the anti-parallel nucleic acid single strands,thereby forming a double-stranded nucleic acid between the complementarystrands.

The terms “first” and “second” strand refer to the strands of adouble-stranded nucleic acid, where one strand can be regarded as thefirst strand, and its complementary strand can be regarded as the secondstrand. Alternatively, the two nucleic acid strands of thedouble-stranded nucleic acid may be referred to as the 5′ to 3′ strandand its complement, the 3′ to 5′ strand.

As used herein, “positive” or “sense” strand refers to the strand of theDNA duplex of a gene that contains the sequence of the correspondingmRNA transcript of the gene.

“Negative strand” or “antisense” strand refers to the strand of the DNAduplex of a gene that contains the sequence that is complementary to thecorresponding mRNA transcript of the gene.

A “target” nucleic acid or sample as used herein refers to the nucleicacid used for analysis and to which a polynucleotide for HSV detectionor for an internal amplification control and/or a pair of PCR primers ishybridized in order to ascertain the presence or absence of the nucleicacid.

As used herein, “forward amplification primer” refers to apolynucleotide used for PCR amplification that is complementary to thesense strand of the target nucleic acid. “Reverse amplification primer”refers to a polynucleotide used for PCR amplification that iscomplementary to the antisense strand of the target nucleic acid. For agiven target, a forward and reverse amplification primer are used toamplify the DNA in PCR.

As used herein, “oligonucleotide primers” refer to single-stranded DNAor RNA molecules that are capable of hybridizing to a nucleic acidtemplate and are capable of priming (or initiating) enzymatic synthesisof a second nucleic acid strand.

As used herein, “amplifying” refers to producing additional copies of anucleic acid sequence, preferably by the method of polymerase chainreaction (Mullis and Faloona, 1987, Methods Enzymol., 155:335)

“PCR product” as used herein refers to the nucleic acid generated fromPCR amplification of a given region of a target nucleic acid.

“Hybrid” as used herein refers to a double-stranded nucleic acid, or aregion having a double-stranded nucleic acid, in which the first andsecond strands of the nucleic acid are complementary to each other.

A “sample” as used herein refers to a target nucleic acid, and mayconsist of purified or isolated nucleic acid, or may comprise abiological sample such as a tissue sample, a biological fluid sample, acell sample containing a nucleic acid.

“Template” as used herein refers to a nucleic acid sequence thatencompasses the region of the target sequence to which thepolynucleotides and primers are complementary.

“Control DNA template” in general as used herein refers to thesequence-matched targets useful in the invention.

“Internal amplification control” or “IAC” as used herein serves todetect false negatives and/or false positives in the assay for HSVdetection. The IAC comprises a control plasmid that contains sequencescapable of being amplified by the same PCR primers as those used for HSVsequence amplification and sequences to which a polynucleotide ormolecular beacon is capable of hybridizing but to which a polynucleotideor molecular beacon for HSV detection cannot bind, and a controlsingle-stranded polynucleotide target to which the IAC molecular beaconcan hybridize.

Polynucleotides

All polynucleotides, including PCR amplification primers andsingle-stranded molecular beacon targets, were custom synthesized atBioCrest (Bastrop, Tex.). Molecular beacons were custom synthesized andpurified at Midland Certified Reagent Company (Midland, Tex.). PCRprimer design and target hybridization temperature estimations werebased on the HSV type-1 and type-2 nucleic acid sequence and aided bythe Oligo 5.0 program for a PC (MBI, Cascade, Colo.). Molecular beaconfolding patterns and stem melting temperatures were approximated by afolding program provided by Washington University, St. Louis, Mo.(http://mfold1.wustl.edu/˜mfold/dna/form1.cgi).

Design of Molecular Beacons for HSV Detection, HSV Type Discriminationand IAC

The present invention allows the simultaneous use of two molecularbeacons in the same reaction. The molecular beacons can differ only by asingle nucleotide, therefore not only enabling the detection of HSVgenerally in a sample but also enabling the discrimination of thespecific HSV type in the sample. It also definitively discriminates atrue negative result from a false negative result that is due to PCRfailure by the use of an internal amplification control in the samereaction. Therefore, the molecular beacons of the present invention areparticularly suitable as hybridization probes for HSV detection and forHSV type discrimination.

For detection of HSV, a single region which could be used for both atype common (TC) and a type specific assay is selected. In other words,a region which contains both identity and variation between the twoviral types is desirable. After comparing a number of type 1 and type 2genomes, a region within the gB gene is selected. The gB region containsa number of nucleotide differences between the two types (all C to Gchanges), but also contains a single T to C polymorphism (FIG. 1). HSV-1contains a C and HSV-2 contains a T base. The molecular beacon for thetype common assay (HSV-TC molecular beacon) is centered on that T/Cnucleotide and used the universal K base at that location within thebeacon (Table 1). The K base has been shown to hybridize with either a Tor a C (Hill, et al., 1998a and Hill, et al., 1998b), and thereforeallows for the detection of either viral type.

For discrimination between HSV type-1 and type-2, the same region isused for molecular beacons containing either a T or a C to differentiatebetween the viral types (Table 1).

The IAC sequence is simply a region of the HSV sequence but in theopposite orientation. An IAC sequence encompasses the same amplificationprimer binding sites as those used to amplify the HSV sequence. TheIAC-specific molecular beacon is shown in Table 1. Both the HSVmolecular beacons and IAC-specific molecular beacons hybridize to thesense strand.

As described, the present invention allows simultaneous use of more thanone molecular beacon. When more than one molecular beacon is used, theyare preferably labeled with different fluorophores that emit fluorescentlight at specific optical wavelengths. For example, the HSV-TC molecularbeacon can be simultaneously used with the IAC molecular beacon. As aresult, detection of HSV can be carried out in the same PCR reaction asa control reaction. In most cases, the fluorophore TET(Tetrachloro-fluorescein) is used to label the HSV molecular beacons andFAM (6-carboxy--fluorescein) is used to label the IAC-specific beacon.When discriminating between HSV types in the same reaction, the HSVmolecular beacon specific for one type may be labeled with TET while theHSV molecular beacon specific for the other HSV type is labeled withFAM. The two fluorescent signals can be distinguished by using theABI7700 sequence detector software. DABCYL is used as the quencher forall of the molecular beacons. Table 1 lists the sequences of themolecular beacons used in the kits. These beacons are dissolved in TEbuffer (10 mM Tris and 1 mM EDTA, pH 8.0) and stored at −20° C. Theconcentrations of all the beacons are determined by UV absorbance (260nm) using a spectrophotometer (Beckman DU600).

TABLE 1 SEQ ID NO POLYNUCLEOTIDE SEQUENCE 1 Forward PCR primer 5′-tcacca ccg tca gca cct tc-3′ 2 Reverse PCR primer 5′-agc agg ccg ctg tccttg-3′ 3 IAC specific molecular beacon 5′-ccctgc gtagtggtacgacctcctgcaggg-3′ 4 HSV type common (TC) 5′-ccctgca aactcgtgKtcctccagcatgcaggg-3′ molecular beacon 5 ssOligonucleotide target for 5′-agc actagg agg tcg tac cac tac aac tcc-3′ IAC specific molecular beacon 6ssOligonucleotide molecular 5′-aac atc acc atg ctg gag gaT cac gag tttgtc ccc ctg-3′ beacon target for HSV type-1 7 SsOligonucleotidemolecular 5′-aac atc acc atg ctg gag gaC cac gag ttt gtc ccc ctg-3′beacon target for HSV type-2 8 Forward primer for synthesis5′-atcgaattctcaccaccgtcagcaccttctagagcaccgcccacatgtggaggt of IACccccctgtttgagcactagg-3′ 9 Reverse primer for synthesis of5′-atcgaattcagcaggccgctgtccttgtagctggagttgtagtggtacgacct IACcctagtgctcaaacaggggg-3′ 10 HSV-1 type specific molecular 5′-ccctgcaaactcgtgTtcctccagca tgcaggg-3′ beacon 11 HSV-2 type specific molecular5′-ccctgca aactcgtgCtcctccagca tgcaggg-3′ beaconInternal Amplification Control (IAC) Plasmid

An internal amplification control is needed in PCR-based assays wherehuman samples (e.g. cerebrospinal fluid (CSF)) usually have a highprobability of containing inhibitors. The internal amplification control(IAC) for the HSV assay is generated by overlap extension of twooligonucleotides, each of which contains one of the PCR amplificationprimer binding regions (see Table 1). Additionally, EcoRI sites areplaced 5′ on each extension oligo in order to facilitate cloning. Theextension is performed with Pfuturbo using the following extensionprofile: 95° C. for 15 sec (1×), followed by 60° C. for 40 sec (40×).The product is gel purified, EtOH precipitated, digested with EcoRI andcloned into pBluescript. The plasmid is sequenced to verify that theproduct contained the IAC sequence.

Assay for HSV Detection

The present invention provides a pair of polynucleotides for polymerasechain reaction (PCR) to amplify a HSV sequence present in a sample. Thepresent invention also provides molecular beacons for detecting both HSVtype-1 and type-2 viral strains. The present invention further providespolynucleotides, including templates and molecular beacons, for internalamplification control reactions to detect false positive and falsenegative results.

Ideally, the PCR primers and molecular beacons hybridize to theirtemplate at the same annealing temperatures so that the PCR anddetection reactions can be performed in the same tube under the same PCRconditions. Upon amplification of the template present in a sample, themolecular beacon hybridizes to the amplified sequence and fluoresces.The fluorescence is detected and is a measure of the quantity of HSVtemplate present in the sample.

Presence of a target or template in the sample is detected by convertingthe emitted fluorescent signal into a threshold cycle (Ct) value.Threshold cycle is the PCR cycle at which the fluorescent signal isfirst detected above background. The Ct values are obtained by analyzingth e fluorescence data using the ABI7700 sequence detection software.The amount of target or template in a sample can be determined byplotting the Ct value generated by the known quantities of a target ortemplate against the Ct value generated by the sample using the samemolecular beacons. In the current study, Ct values of 38-40 (40 is themaximum PCR cycle number used) indicate the absence of a specific targetor template in a sample. Ct values ranging from 20-30 indicate thepresence of a specific target or template in a sample.

Kits

Kits according to the present invention may thus include a pair ofmolecular beacons (a molecular beacon for HSV-TC and another for IAC)and/or a pair of PCR primers having sequences disclosed herein.Optionally, a kit useful according to the invention may also contain oneor more of an HSV type specific molecular beacon which will recognize aspecific HSV type but not the other. The kit according to the inventionmay also contain dNTPs, a DNA polymerase, for example, the Taq2000 DNApolymerase, and a PCR buffer.

The following Examples are intended to illustrate the present invention,but are not intended to be limiting in any manner.

EXAMPLE 1 Optimization of PCR Amplification Conditions

Optimal PCR conditions were determined using the amplification primerswith either the HSV type-1 or HSV type-2 genomic DNA (10,000 copies/rxn)purchased as viral stocks from Advanced Biotechnologies Incorporated(Columbia, Md.). The viral stocks were diluted in HSV negativecerebrospinal fluid (CSF) and stored at −80° C. HSV-negativecerebrospinal fluid (CSF) was obtained from Cenetron Diagnostics bypooling normal patient samples. The PCR buffer used was the molecularbeacons core buffer (1×=70 mM Tris-HCl (pH8.5), 40 mM KCl and 0.1%Tween20). PCR annealing temperature optimization was initiallydetermined using the gradient feature of the RoboCycler 96 (Stratagene).The RoboCycler gradient PCR profile was: 95° C. for 3 min (1×) followedby 95° C. for 15 sec, 55° C.-63° C. gradient for 30 sec, then 72° C. for30 sec (40×) and concluded with a final extension of 72° C. for 5 min(1×). The resulting PCR products were fractionated on 2% (1×TBE) agarosegels and optimal conditions were determined.

FIG. 2 shows that amplification proceeds well at temperatures between55° C. and 63° C. Additionally, non-specific amplification products werenot detected within that annealing range.

EXAMPLE 2 Melting Curve Analysis

A melting curve analysis is carried out by incubating a molecular beaconwith or without its complementary single stranded oligonucleotidetarget. Molecular beacon melting curve analysis was performed todetermine if the HSV molecular beacons are capable of hybridizing to thecorrect targets at the amplification annealing temperature. For the HSVtype common specific beacon (containing the K base), two single-stranded(ss) oligonucleotide targets were evaluated. One ss oligo targetcontained the HSV type 1 sequence (containing a C base), while the othercontained HSV type 2 sequence (containing a T base). For the IACspecific molecular beacon, a single ss oligo target was tested. Thesingle stranded oligonucleotide targets (HSV-1, HSV-2, and IAC) arelisted in Table 1.

FIG. 3 shows the melting curve analysis for the HSV-TC specific and IACspecific molecular beacons. The analysis of each beacon was performed in1×PCR buffer from 90° C. to 20° C., decreasing 1° C./cycle (1 minute). Apotential annealing temperature of 55° C. is indicated. The data showthat at 55° C., both the HSV-TC specific and IAC-specific molecularbeacons form thermally stable complexes with their respective targets,as indicated by the increase in reporter dye fluorescence compared tothe fluorescence generated without target present. Also, the HSV-TCspecific molecular beacon shows no differential hybridization betweenthe HSV-1 and HSV-2 ss oligos, indicating the potential to detect eitherviral template.

EXAMPLE 3 PCR-Molecular Beacon Assay for Detection of HSV Using HSVGenomic DNA

PCR and detection of HSV genomic DNA and IAC plasmid DNA was performedusing the ABI7000. Table 2 shows the PCR conditions used for theamplification and detection of templates. The overall time required forperforming the HSV type common assay is 2 hours and 15 minutes using athree-step amplification profile.

TABLE 2 Final Components Volume Added Concentration PCR buffer (10X)   5μL 1X^(a) dNTPs (20 mM, 5 mM each) 0.5 μL 200 nM Primer mix (20 μM, 10μM each)   2 μL 800 Nm Molecular beacons (MB) mix (10 μM)^(b)   2 μL 400nM^(c) Taq2000 (5 U/μL) 0.5 μL 0.05 U/μL Template variable^(d) variabledH₂O to total volume of 50 μL ABI7700 Amplification Profile: Profile A: 1 cycle 95° C. - 10 min 40 cycles 95° C. - 30 sec/55° C. - 30 sec/72° -30 sec Profile B:  1 cycle 95° C. - 3 min 40 cycles 95° C. - 30 sec/55°C. - 30 sec/72° - 30 sec ^(a)1X = core buffer ^(b)5 μM FAM-labeled IACMB + 5 μM TET-labeled HSV type common (TC) MB ^(c)FAM-labeled IAC MB(200 nM) + TET-labeled HSV MB (200 nM) ^(d)HSV genomic DNA = 0-5000copies per reaction IAC plasmid DNA control = 0-5 pg per reaction

Using the above conditions, the molecular beacons were individuallyexamined for specific hybridization to their respective targets. Themolecular beacon specific for IAC, b290, is labeled with FAM. Themolecular beacon specific for HSV-TC, b294, is labeled with TET. Twoidentical PCR reactions, with the exception of the molecular beaconused, containing IAC as template were evaluated (FIG. 4A). The resultsshow that only b290 (FAM) shows any increase in signal due toamplification of IAC. The b294, specific to the HSV-TC sequence, showedno hybridization. Both reactions showed strong amplification productbands on a gel (data not shown), indicating that the amplification wassuccessful. Similarly, reactions containing only HSV genomic DNA (type-1or type-2) showed the opposite hybridization pattern. Only b294 showedan increase in signal with the generation of HSV-specific amplification(data not shown).

Additionally, two concentrations of HSV-1 and HSV-2 genomic DNA wereevaluated in both the TET and FAM layers for specificity. FIG. 4B showsthe results for HSV-1, while FIG. 4C shows the results for HSV-2. Thedata show that (1) a single molecular beacon with the incorporated Kbase will hybridize to either target with the same efficiency and (2)the associated increase in fluorescent signal is only seen in the TETview (i.e., no signal in the FAM view).

Lastly, FIG. 4D shows two multiplex reactions, one containing HSV-1genomic DNA+IAC plasmid as templates and the other containing HSV-2genomic DNA+IAC plasmid as templates, and a no template control (NTC).All three reactions contained both molecular beacons (b290=IAC specificand b294=HSV-TC specific). The amplification plots show increases inboth the FAM and TET views indicating the presence of both templates.This was the first demonstration that multiplexes reactions for thesimultaneous detection of herpes simplex (either type) and the internalamplification control would work.

Likewise, HSV type specific molecular beacons representing SEQ ID NO: 10and SEQ ID NO:11 can also be used in the same reaction in order toverify which type of HSV is present in the sample. For example, tworeactions can be set up, one containing HSV-1 genomic DNA and, inanother tube, HSV-2 genomic DNA wherein both tubes contain both typespecific molecular beacons. The molecular beacons are preferablydifferentially labeled, for example, HSV-1 type specific beacon islabeled with TET and HSV-2 type specific beacon is labeled with FAM. Inthis example, the amplification plots would show a TET signal if HSVtype-1 is present in the sample and a FAM signal if HSV type-2 ispresent in the sample. In this manner, this method can be used for thediscrimination of HSV strain types.

Alternatively, the HSV type specific molecular beacons can be usedseparately but simultaneously with IAC. For example, HSV type-1 specificmolecular beacon can be used with the IAC in the same reaction in onetube and the HSV type-2 specific molecular beacon can be used with theIAC in a separate reaction in another tube. In this embodiment, the HSVtype specific molecular beacons are preferably differentially labeledfrom the IAC specific molecular beacon. For example, both the HSV type-1and type-2 molecular beacons can be labeled with TET while the IACspecific molecular beacon is labeled with FAM. If a specific HSV type ispresent in a sample, one tube will emit TET while the other tube willnot emit TET. If both tubes contain an IAC plasmid, both tubes shouldemit FAM if the reaction has proceeded properly. This method can be usedas an alternative method for discriminating between HSV strain types.

EXAMPLE 4 Sensitivity of HSV-TC (K Base) Molecular Beacon

The sensitivity of HSV detection was compared between the PCR assay ofthe present invention and the standard microtiter plate assay. Bothassays used dilutions of HSV-1 and HSV-2 obtained from commercial HSVDNA standards (ABI, Inc., Columbia, Md.). Serial dilutions of HSV-1 andHSV-2 (10⁵ copies to 10⁰ copies per reaction) were tested with theHSV-TC molecular beacon. FIG. 5 shows that both HSV-1 (panel A) andHSV-2 (panel B) are easily detected down to 100 copies/reaction. Greatersensitivity is often seen, such as the 10¹ and 10⁰ samples of HSV-1 inthis data. However, the reliable detection limit for this assay is 100copies per reaction of either HSV-1 or HSV-2.

The HSV-1 and HSV-2 serial dilutions were additionally tested using astandard microtiter plate assay format (Cenetron Diagnostics SOP 6000.1)for the detection of HSV and an internal control (called ICC forinternal control cassette, WalkerPeach and DuBois, patent pending).Table 3 shows the comparison of the data from the two assay formats. ThePCR assay of the present invention, using HSV-1 as template, appears 5×more sensitive than the plate-based assay, whereas a greater differenceis seen when using HSV-2 as template (5× to 50× more sensitive thanplate-based assay).

TABLE 3 MICROTITER PLATE ASSAY REAL-TIME MULTIPLEX ASSAY Dilution OD405OD405 Ct Ct Sample (copies/rxn) (HSV) (ICC) Result (Tet-HSV) (Fam-IAC)Result HSV-1 10^5 2.97 1.55 Positive 23.20 26.79 Positive HSV-1 10^42.35 1.36 Positive 25.94 27.24 Positive HSV-1 10^3 0.80 1.31 Positive27.74 26.75 Positive HSV-1 10^2 0.20 1.84 Negative 32.05 27.50 PositiveHSV-1 10^1 0.18 1.29 Negative 34.63 27.81 Negative HSV-2 10^5 2.60 1.13Positive 23.94 27.37 Positive HSV-2 10^4 1.65 1.08 Positive 27.16 27.76Positive HSV-2 10^3 0.27 1.32 Negative 29.82 28.45 Positive HSV-2 10^20.23 1.81 Negative 32.85 28.81 Positive HSV-2 10^1 0.18 1.60 Negative37.85 29.48 Negative NTC — 0.16 2.11 Negative 40.00 27.50 Negative

EXAMPLE 5 HSV Detection in Clinical Samples

Ten (10) CSF specimens (HSV status unknown) were evaluated with thePCR-molecular beacon assay. The CSF specimens were prepared by threemethods (1) Cenetron Diagnostics SOP 6000.1, (2) Qiagen blood kit asmodified for CSF and (3) no preparation (i.e. direct incubation withmaster mix). The data are not shown for preparation (1) and (2), due tofailure of amplification and/or detection. It is hypothesized that inthe case of (1), the concentrations of detergent (NP-40) and reducingagent (DTT) caused the inhibition of detection (bands seen on a gel). Inthe case of (2), it is believed that after the isolation procedure wascompleted, none or very little, of the HSV DNA remained, thereby causinga failure in amplification (no bands seen on gel). The third method of‘no preparation’ of the specimens gave the best data and can be seen inFIG. 6.

The 10 ‘no prep’ samples were run along side two positive controls (10⁵and 10² copies of HSV-1 per reaction) and a negative (CSF). Specimens 2,3, 5, 6, 9 and 10 were negative. Three specimens were positive, (4 and 8(roughly 10⁵ copies/mL of CSF)), followed by specimen 1 (approximately10⁴ copies/mL CSF). Specimen 7 gave a Ct value of 38 indicating a verylow positive (<10² copies/mL of CSF). The results were in 100%concordance with the results performed at Cenetron Diagnostics using amicrotiter-based detection system.

Although the ‘no prep’ method significantly reduces pre-amplificationmanipulation steps, the level of Ct variation in the clinical samplesobtained for IAC is fairly high (FIG. 6, lower amplification plot). Thisvariation is likely attributed to the normal variation of amplificationinhibitors, protein, heme and other components found in individualpatient CSF specimens. This illustrates the importance for an IAC in themaster mix to detect false positives and false negatives.

To eliminate the variation associated with IAC, a second ‘no prep’method was evaluated and compared. As discussed above, the methodologyfor clinical specimen preparation involves directly adding 25 μL of CSFto 25 μL of master mix (herein after “Method 3”). In this second ‘noprep’ method (herein after “Method 3A”), the amplification profile waspreceded by an incubation step of 95° C. for 10 min in the thermocycler.During the incubation, the HSV capsid structure, as well as otherproteins found in the CSF, is most likely to become disassociatedthereby exposing the template DNA to the amplification process.

Three CSF patient samples were obtained from Cenetron Diagnostics (CedarCreek, Tex.) that allowed duplicate samples to be run in two separateexperiments. Two of the specimens were negative on the microtiter plateassay, while the third was positive (data not shown). These specimenswere used to compare Method 3 with Method 3A. Results using Method 3 areshown in FIG. 7. In Method 3A, 35 μL of each specimen to be tested washeated to 95° C. for 10-15 min, centrifuged and then a 25 μL aliquot wasremoved and added to the master mix (FIG. 8), followed by thePCR-molecular beacon assay of the present invention. Method 3A showsless variability in the IAC, suggesting that the pre-incubation step canalso reduce variability in detection of HSV.

Following the initial HSV detection in the samples, the samples can befurther tested for determination of which type of HSV is present in thesample. HSV type discrimination can be carried out by either using theHSV type specific molecular beacons separately or by simultaneouslyusing both HSV type specific molecular beacons in the same reaction asdescribed in Example 4.

EXAMPLE 6 Kits for HSV Detection

Examples of kits useful for approximately 100 reactions are shown inTables 4-7.

TABLE 4^(a) Material Quantity 10X PCR buffer^(b) 500 μL 20 mM dNTPs^(c) 50 μL 30 μM molecular beacons mix^(d) 175 μL 40 μM primer mix^(e) 300μL  5 U/μL Taq2000  50 μL HSV Type-specific controls^(f): HSV Type-1(10⁵ copies/rxn) 250 μL HSV Type-1 (10² copies/rxn) 250 μL HSV Type-2(10⁵ copies/rxn) 250 μL HSV Type-2 (10² copies/rxn) 250 μL^(a)Components for 100 assays per kit (50 uL per assay reaction volume)includes 40 type-specific control reactions ^(b)10X = Core buffer (700mM Tris, 400 mM KCl, 1% Tween) + 30 mM MgCl₂ (pH 8.5) ^(c)5 mM each dNTP^(d)20 μM FAM-labeled IAC-specific MB + 10 μM TET-labeled HSV-TC MB^(e)20 μM each primer + IAC plasmid (83.3 pg/mL, 75,000 copies/rxn)^(f)10⁵ = 4000 copies/μL, 10² = 4 copies/μL

TABLE 5^(a) Material Quantity 10X PCR buffer^(b) 500 μL 20 mM dNTPs^(c) 50 μL 30 μM molecular beacons mix^(d) 175 μL 40 μM primer mix^(e) 300μL  5 U/μL Taq2000  50 μL HSV Type-specific controls^(f): HSV Type-1(10⁵ copies/rxn) 250 μL HSV Type-1 (10² copies/rxn) 250 μL HSV Type-2(10⁵ copies/rxn) 250 μL HSV Type-2 (10² copies/rxn) 250 μL^(a)Components for 100 assays per kit (50 uL per assay reaction volume)includes 40 type-specific control reactions ^(b)10X = Core buffer (700mM Tris, 400 mM KCl, 1% Tween) + 30 mM MgCl₂ (pH 8.5) ^(c)5 mM each dNTP^(d)20 μM FAM-labeled IAC-specific MB + 10 μM TET-labeled HSV type-1specific MB ^(e)20 μM each primer + IAC plasmid (83.3 pg/mL, 75,000copies/rxn) ^(f)10⁵ = 4000 copies/μL, 10² = 4 copies/μL

TABLE 6 Material Quantity 10X PCR buffer^(b) 500 μL 20 mM dNTPs^(c)  50μL 30 μM molecular beacons mix^(d) 175 μL 40 μM primer mix^(e) 300 μL  5U/μL Taq2000  50 μL HSV Type-specific controls^(f): HSV Type-1 (10⁵copies/rxn) 250 μL HSV Type-1 (10² copies/rxn) 250 μL HSV Type-2 (10⁵copies/rxn) 250 μL HSV Type-2 (10² copies/rxn) 250 μL ^(a)Components for100 assays per kit (50 uL per assay reaction volume) includes 40type-specific control reactions ^(b)10X = Core buffer (700 mM Tris, 400mM KCl, 1% Tween) + 30 mM MgCl₂ (pH 8.5) ^(c)5 mM each dNTP ^(d)20 μMFAM-labeled IAC-specific MB + 10 μM TET-labeled HSV type-2 specific MB^(e)20 μM each primer + IAC plasmid (83.3 pg/mL, 75,000 copies/rxn)^(f)10⁵ = 4000 copies/μL, 10² = 4 copies/μL

TABLE 7^(a) Material Quantity 10X PCR buffer^(b) 500 μL 20 mM dNTPs^(c) 50 μL 20 μM molecular beacons mix^(d) 175 μL 40 μM primer mix^(e) 300μL  5 U/μL Taq2000  50 μL HSV Type-specific controls^(f): HSV Type-1(10⁵ copies/rxn) 250 μL HSV Type-1 (10² copies/rxn) 250 μL HSV Type-2(10⁵ copies/rxn) 250 μL HSV Type-2 (10² copies/rxn) 250 μL^(a)Components for 100 assays per kit (50 uL per assay reaction volume)includes 40 type-specific control reactions ^(b)10X = Core buffer (700mM Tris, 400 mM KCl, 1% Tween) + 30 mM MgCl₂ (pH 8.5) ^(c)5 mM each dNTP^(d)10 μM FAM-labeled HSV type-2-specific MB + 10 μM TET-labeled HSVtype-1 specific MB ^(e)20 μM each primer (83.3 pg/mL) ^(f)10⁵ = 4000copies/μL, 10² = 4 copies/μL

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed polynucleotides,methods and kits. Variation and changes are intended to be within thescope and nature of the invention which is defined by the appendedclaims.

1. A polynucleotide for HSV detection, wherein said polynucleotidecomprises SEQ ID NO:4.
 2. The polynucleotide of claim 1 wherein thepolynucleotide comprises a labeled polynucleotide.
 3. The polynucleotideof claim 2 wherein the labeled polynucleotide comprises a pair offluorophore/quencher labels.
 4. A pair of polynucleotides for HSVdetection wherein said pair of polynucleotides is selected from thegroup consisting of SEQ ID NO:3 and SEQ ID NO:4, and SEQ ID NO:3 and,SEQ ID NO:3.
 5. The pair of polynucleotides of claim 4 wherein said pairof polynucleotides comprises a labeled first polynucleotide and alabeled second polynucleotide, wherein the first and secondpolynucleotides are differently labeled.
 6. The pair of polynucleotidesof claim 5 wherein said first and second differently labeledpolynucleotides each comprises a pair of fluorophore/quencher labels. 7.The pair of polynucleotides of claim 6 wherein said fluorophore label isdifferent between said first and second polynucleotides and wherein saidquencher label is the same between first and second polynucleotides. 8.A kit for HSV detection comprising a pair of polynucleotides of claim 4and packaging materials therefor.
 9. The kit of claim 8 furthercomprising a pair of polynucleotide primers for a polymerase chainreaction, wherein the primers comprise SEQ ID NO:1 and SEQ ID NO:2, anda DNA polymerase.
 10. A kit for performing a polymerase chain reactioncomprising a pair of polynucleotide primers of claim 4, a DNApolymerase, and packaging materials therefor.
 11. The kit of claim 9 or10 wherein said DNA polymerase is thermostable.
 12. The kit of claim 8or 10 further comprising a buffer suitable for HSV detection andpolymerase chain reaction.
 13. The kit of claim 12 further comprising aninternal amplification control plasmid comprising sequences presented inSEQ ID NO:8 AND SEQ ID NO:9.
 14. The kit of claim 13 further comprisinga first control template having a sequence presented in SEQ ID NO:6 anda second control template having a sequence presented in SEQ ID NO:7.15. A kit for HSV detection, comprising a polynucleotide for HSVdetection having a sequence presented in SEQ ID NO:4, a pair ofpolynucleotides for polymerase chain reaction wherein a fistpolynucleotide of said pair has the sequence presented in SEQ ID NO:1and a second polynucleotide of said pair has the sequence presented inSEQ ID NO:2, a DNA polymerase, and a buffer suitable for HSV detectionand polymerase chain reaction.
 16. The kit of claim 15 furthercomprising a control polynucleotide having a sequence presented in SEQID NO:3, and an IAC plasmid comprising sequences presented in SEQ IDNO:8 AND SEQ ID NO:9.
 17. The kit of claim 15 or 16 further comprising afirst control template having a sequence presented in SEQ ID NO:6 and asecond control template having a sequence presented in SEQ ID NO:7. 18.A kit for HSV detection, comprising a pair of polynucleotides for HSVdetection selected from the group consisting of SEQ ID NO:3 and SEQ IDNO:4, or SEQ ID NO:3 and SEQ ID NO:3, a pair of polynucleotides forpolymerase chain reaction wherein a first polynucleotide of said pairfor polymerase chain reaction has the sequence presented in SEQ ID NO:1and a second polynucleotide of said pair for polymerase chain reactionhas the sequence presented in SEQ ID NO:2, a DNA polymerase, and abuffer suitable for HSV detection and polymerase chain reaction.
 19. Thekit of claim 18 further comprising a control polynucleotide having asequence presented in SEQ ID NO:3, and an IAC plasmid comprisingsequences presented in SEQ ID NO:8 AND SEQ ID NO:9.
 20. The kit of claim18 or 19 further comprising a first control template having a sequencepresented in SEQ ID NO:6 and a second control template having a sequencepresented in SEQ ID NO:7.